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Old 06-05-2014, 11:35 PM   #1
ZanderH22
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Location: Pretoria, RSA

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Default Best 16S region for 300PE Miseq

Hi,

I was wondering if anyone has any thoughts on the most reliable variable regions for 16S rRNA sequencing on Miseq with the 300bp PE reagents. It would be ideal to me to do V1-3, but I've heard that it would be better going for a region with a bigger overlap ie. V3-4 since error correcting is harder with short overlaps. Any thoughts?
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Old 03-01-2015, 01:10 PM   #2
MicroBio
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Sorry this is a super late response, but maybe it can help somebody in the future.

It can really depend on the kinds of bacteria you plan on finding. Different variable regions are better for discriminating between different bacteria. I would look into the literature to see what regions are thought to be best for your community of interest.
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Old 03-13-2015, 08:39 AM   #3
lorendarith
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I believe that you'll gain very little by doing 2x300bp instead of 2x250bp on the MiSeq for 16S amplicon data, or at least it used to be like this a year ago. The quality of the last 50-100bp of 300bp reads can vary quite a lot and is usually very low, so unless you have completely overlapping reads you won't really benefit from these additional bases if you used 600cycles instead of the 500cycles kit.

Take a look at these posts for more info:
http://www.mothur.org/forum/viewtopi...e25054457c20cf
http://blog.mothur.org/2014/09/11/Wh...nce-matrix%3F/
pschloss.github.io/talks/2014_08_28_illumina
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